Antibodies are made up of two distinct regions, referred to as the variable (Fv) and constant (Fc) regions. The Fc region of an antibody interacts with a number of ligands, such as Fc receptors and C1q, imparting an array of functional capabilities referred to as effector functions. Fc receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., Annu. Rev. Cell. Dev. Biol. 12:181-220 (1996); Ravetch et al., Annu. Rev. Immunol. 19:275-290, (2001)).
The formation of the Fc/FcγR complex typically resulting in signaling events within these cells and subsequent immune responses such as release of inflammation mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize an antibody bound on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Ghetie et al., Annu. Rev. Immunol. 18:739-766 (2000); Ravetch et al., Annu. Rev. Immunol. 19:275-290 (2001)).
Human FcγRs are divided into three distinct classes: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). IgG molecules exhibit differential isotype specificity for FcγRs. IgG3 molecules bind strongly to all FcγR isoforms. IgG1, the most prevalent isoform in the blood binds to all FcγRs albeit with a lower affinity for the FcγRIIA/β isoforms. IgG4 is an intermediate binder to FcγRI and a weak binder to FcγRIIB. Finally, IgG2 binds only weakly to one allelic form of FcγRIIA (FcγRIIA-H131) (Siberil et al., J. Immunol. Lett. 106:111-118 (2006)). A short continuous stretch of amino acid residues (234-238) of the N-terminus part of the CH2 region as being directly involved in the binding to all FcγRs. Residues 268, 297, 327 and 329 can also impact binding to a subset of FcγRs, and multiple residues located in the CH2 and CH3 regions also contribute to FcγR binding (Canfield et al., J. Exp. Med. 173:1483-91 (1991), Chappel et al., Proc. Natl. Acad. Sci. USA 888:9036-40 (1991), Gergely et al. FASEB J. 4:3275-83 (1990)).
An overlapping site on the Fc region of the molecule controls the activation of a cell independent cytotoxic function mediated by complement. Accordingly, Fc binding to complement protein C1q mediates a process called complement dependent cytotoxicity (CDC) (see Ward et al., Ther. Immunol. 2:77-94 (1995)).
In certain instances it is advantageous to decrease or eliminate effector function. In these cases the use of antibodies or Fc domain-containing fragments that poorly recruit complement or effector cells is beneficial (see, e.g., Wu et al., Cell Immunol. 200:16-26 (2000); Shields et al., J. Biol. Chem. 276:6591-6604 (2001); U.S. Pat. No. 6,194,551; U.S. Pat. No. 5,885,573; PCT publication WO 04/029207; and U.S. Publ. No. 2011/0059078).
Although certain subclasses of human immunoglobulins poorly recruit complement or effector cells, for example IgG2 and IgG4, there are no known naturally occurring immunoglobulins that lack all effector functions. Thus, an alternate approach is to engineer or mutate residues in the Fc region that are responsible for effector function. See, e.g., PCT publications WO2006076594, WO199958572, WO2006047350, and WO2006053301; U.S. Pat. Pub. No. 2006-0134709; U.S. Pat. Nos. 5,624,821, 6,194,551, and 5,885,573; Armour et al., Eur. J. Immunol. 29:2613-2624 (1999); Reddy et al., J. Immunol. 164:1925-1933 (2000); Xu et al., Cell Immunol. 200:16-26 (2000); Shields et al., J. Biol. Chem. 276:6591-6604 (2001).
A consideration for the reduction or elimination of effector function is that other important antibody properties not be perturbed. Thus, Fc variants should be engineered to only ablate binding to FcγRs and/or C1q, while maintaining antibody stability, solubility, and structural integrity, as well as the ability to interact with other important Fc ligands such as FcRn and proteins A and G.